Field of Invention
This invention concerns streptavidin-metallothionein chimeric proteins which possess biological recognition specificity.
Recombinant streptavidin-metallothionein chimeric proteins containing various metal ions are molecules which have a great potential in preventive and therapeutic medicine in both humans and animals as well as for diagnostic use. While each individual molecule, i.e., streptavidin and metallothionein have been known and described previously, their composite molecule has never before been constructed.
Biochemistry of metallothionein, particularly its amino acid sequence in various species, its metal binding sites, metal thiolate clusters and spatial structures are described in Biochemistry, 27:509 (1988). Ann. Rev. Biochem., 55:913 (1986) is directed to the gene structure, organization, amplification and transcriptional regulations, and describes also some genetic engineering applications, such as for example, metallothionein-rat growth hormone and metallothionein-human growth hormone genes expressed in transgenic mice, or conferring resistance to copper toxicity via the CUP 1 copper-metallothionein gene.
Streptavidin is a protein very closely related to a protein avidin which provides a very stable noncovalent complex with vitamin D-biotin. Avidin itself is a very highly specialized protein that is only rarely expressed. Streptavidin, on the other hand, is readily expressed in Streptomyces species particularly in Streptomyces avidinii. Streptavidin specifically binds a water soluble vitamin D-biotin (vitamin H). Similarly to avidin, it binds rapidly and almost irreversibly to any molecule which contains unhindered biotin with a remarkably high affinity. Streptavidin, contrary to avidin, is carbohydrate free and thus more suitable, for example, for X-ray crystallographic studies or for various other detection techniques. The comparative properties of avidins and streptavidins are described in Methods in Enzymology, 184:51 (1990). Isolation and properties of streptavidin, as well as its preparation, are described in Ibid., at page 80.
Expression of a cloned streptavidin gene in Escherichia coli is described in Proc. Natl. Acad. Sci., 87:142 (1990) and the cooperativity in the biotin binding to streptavidin is described in J. Biol. Chem., 265:3369 (1990).
One aspect of the current invention is a recombinant streptavidin-metallothionein chimeric protein having biological recognition specificity.
Another aspect of the current invention is an expression system for the cloned streptavidin gene which expresses streptavidin in Escherichia coli and allows the expression of a streptavidin-metallothionein chimeric protein.
Another aspect of the current invention is an expression vector pTSAMT-2 constructed by inserting the mouse metallothionein-I cDNA into an expression vector for streptavidin-containing chimeric proteins pTSA-I8F.
Another aspect of the current invention is the expression of the gene fusion of streptavidin with metallothionein using T7 expression system.
Still another aspect of the current invention is the method for binding streptavidin-metallothionein chimeric protein with various metal ions.
Still yet another aspect of the current invention is incorporation of the metal-containing streptavidin-metallothionein chimeric protein into biological materials containing unhindered biotin.
Yet another aspect of the current invention is the method for introducing heavy metal ions into the tissue, removing the heavy metal ions from the tissue or labeling the tissue with heavy metal ions.
Still another aspect of the current invention is the use of the streptavidin-metallothionein chimeric protein for imaging of tumors, for radiotherapeutics, for labeling of biological materials, for detection of biological molecules present at very low levels and for simultaneous multi-mass labeling of short DNA molecules allowing determination of a number of DNA sequences.